Project Summary Dihydropyridine-sensitive, L-type Cav1.2 and delayed rectifier Kv2.1 channels play critical roles in the regulation of excitability and contraction in arterial smooth muscle. A salient feature of Cav1.2 channels is that they form clusters within which they undergo dynamic, reciprocal interactions that allow functional coupling of adjacent channels and thus amplification of Ca2+ signaling, which is critical to the development of myogenic tone. At present, however, the mechanisms controlling Cav1.2 clustering are unknown. The Trimmer and Santana labs have joined forces to address this fundamental issue. New preliminary data from our labs suggest a novel model that represents a paradigm shift relative to the generally accepted canonical role of Kv2.1, and K+ channels in general, as acting solely as K+ conducting electrical determinants of the intrinsic membrane properties of arterial myocytes. In this model, the Kv2.1 channel has a physical role to increase clustering and thus cooperative gating of Cav1.2 channels. Our data indicate that the balance between the separable electrical and structural roles of Kv2.1 channels fine tunes membrane potential, Cav1.2 clustering, functional coupling of these channels, and hence Ca2+ influx, myogenic tone, and, ultimately, blood pressure. A key finding that underscores the significance of our work is that Kv2.1 expression varies with sex, leading to significant differences in Ca2+ influx and myogenic tone between female and male arterial myocytes. The combination of our complementary skill sets allows us to implement a multi-scale systems approach that involves the use of cellular, molecular, biophysical, imaging, gene editing and whole-animal approaches to rigorously investigate the mechanisms controlling Kv2.1 and Cav1.2 organization, and how they impact cell, organ, and whole-body functions under physiological conditions. The project has three specific aims. Aim 1 is to determine the impact of altered Kv2.1 expression levels on clustering and activity of Cav1.2 channels, and myogenic tone in arterial smooth muscle, and on blood pressure. Aim 2 is to define the mechanisms underlying Kv2.1-mediated regulation of Cav1.2 function. Finally, Aim 3 is to use novel genetic models to define the cell autonomous role of Kv2.1, and its separable conducting and non-conducting functions, in regulating Cav1.2 function, and the myogenic response in arterial smooth muscle cells, and systemic blood pressure. The proposed studies have the potential of transforming our understanding of how ion channels are organized in vascular smooth muscle, and provide insights into how arterial diameter and blood pressure are differentially regulated in females versus males.